CuAPO-5 as a Multiphase Catalyst for Synthesis of Verbenone from α-Pinene

Copper(II)-containing aluminum phosphate material (CuAPO-5) was synthesized hydrothermally and used as a multiphase catalyst for the oxidation of α-pinene to verbenone. The catalysts were analyzed using X-ray diffraction (XRD), Brunauer–Emmett–Teller (BET) surface area techniques, X-ray photoelectron spectroscopy (XPS), and ammonia temperature programmed reduction (NH₃-TPD). Scanning electron microscopy (SEM), X-ray energy spectrometry (EDS), inductively coupled plasma emission spectroscopy (ICP-OES), Fourier infrared spectroscopy (FT-IR), and ultraviolet-visible spectroscopy (UV-vis) were performed to characterize the material. The effects of reaction temperature, reaction time, n(α-pinene)/n(TBHP), and solvent on the catalytic performance of CuAPO-5 were investigated. The results show that all the prepared catalysts have AFI topology and a large specific surface area. Copper is evenly distributed in the skeleton in a bivalent form. The introduction of copper increases the acid content of the catalyst. Under the optimized reaction conditions, 96.8% conversion of α-pinene and 46.4% selectivity to verbenone were achieved by CuAPO-5(0.06) molecular sieve within a reaction time of 12 h. CuAPO-5(0.06) can be recycled for five cycles without losing the conversion of α-pinene and the selectivity to verbenone.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

Deformation and Phase Transformation of Disordered α Phase in the (α + γ) Two-Phase Region of a High-Nb TiAl Alloy

In this paper, the deformation and phase transformation of disordered α phase in the (α + γ) two-phase region in as-forged Ti-44Al-8Nb-(W, B, Y) alloy were investigated by hot-compression and hot-packed rolling. The detailed microstructural evolution demonstrated that the deformed microstructure was significantly affected by the deformation conditions, and the microstructure differences were mainly due to the use of a lower temperature and strain rate. Finer α grains were formed by the continuous dynamic recrystallization of α lamellae and α grains distributed around lamellar colonies. Moreover, the grooved γ grains formed by the phase transformation from α lamellae during hot rolling cooperated with and decomposed α lamellae. A microstructure evolution model was built for the TiAl alloy at 1250 °C during hot rolling.     

Role of α1‐blockers in the current management of hypertension

There is emerging evidence that α1‐blockers can be safely used in the treatment of hypertension. These drugs can be used in almost all hypertensive patients for blood pressure control. However, there are several special indications. Benign prostatic hyperplasia is a compelling indication of α1‐blockers, because of the dual treatment effect on both high blood pressure and lower urinary tract symptoms. Many patients with resistant hypertension would require α1‐blockers as add‐on therapy. Primary aldosteronism screen is a rapidly increasing clinical demand in the management of hypertension, where α1‐blockers are useful for blood pressure control in the preparation for the measurement of plasma aldosterone and renin. Nonetheless, α1‐blockers have to be used under several considerations. Among the currently available agents, only long‐acting α1‐blockers, such as doxazosin gastrointestinal therapeutic system 4–8 mg daily and terazosin 2–4 mg daily, should be chosen. Orthostatic hypotension is a concern with the use of α1‐blockers especially in the elderly, and requires careful initial bedtime dosing and avoiding overdosing. Fluid retention is potentially also a concern, which may be overcome by combining an α1‐blocker with a diuretic.     

The N terminus of α-synuclein dictates fibril formation

The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson’s disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41–140). This behavior can be explained by the unique fibril structure that is adopted by 41–140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent β-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended β-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.

Amyloid formation of α-synuclein (α-syn) is a pathological feature of Parkinson’s disease (PD), multiple-system atrophy (MSA), and dementia with Lewy bodies (1, 2). An abundant presynaptic protein (3), α-syn is 140 amino acids in length with a putative biological function in aiding the exocytosis of synaptic vesicles (4–6), in which the first 89 N-terminal residues fold into a helical structure upon membrane association (7). In its disease-associated, aggregated amyloid state, residues 37 through 97 adopt β-sheet structure (8), which overlaps with the lipid-binding domain. Notably, both N- and C-terminal α-syn truncations are associated with PD (9). So far, N-terminally truncated (ΔN) α-syn variants 5‒140, 39‒140, 65‒140, 66‒140, 68‒140, and 71‒140 and C-terminally truncated (ΔC) α-syn variants, 1‒101, 1‒103, 1‒115, 1‒122, 1‒124, 1‒135, and 1‒139 have been found in brains of PD patients (10–12).α-Syn truncations originate from incomplete degradation, which has been attributed to various cytosolic (13–15) and lysosomal proteases (16, 17). In fact, ∼60% of the abovementioned truncations can be assigned to cleavages by lysosomal asparagine endopeptidase (AEP), cathepsin (Cts) D, CtsB, and CtsL (15–17). Removal of the C terminus (residues 104–140) is shown to accelerate fibril formation both in vitro and in vivo (18–25). On the other hand, perplexing behaviors of ΔN-variants have been documented; while deleting the first 20 residues has minimal perturbation, the removal of either the first 10 or 30 residues slows aggregation kinetics (26). Nevertheless, the influence of N-terminal residues on α-syn aggregation has been shown by both insertion [tandem repeat of residues 9–30 (27)] and deletion [Δ36–42 (28) and Δ52–55 (29)] mutants, in which fibril formation can be completely impeded.Recently, structure determination by cryogenic electron microscopy (cryo-EM) has revealed fibril structures for full-length α-syn (1–140) (24, 30–32), C-terminal truncations (24, 33), phosphorylated Y39 (34), and PD-related mutants, E46K (35, 36), H50Q (37), and A53T (38). One striking feature of these fibrils is the eclectic mix of structures, often termed as fibril polymorphism. In fact, it was recently shown that different conformational strains of α-syn fibrils are present in PD and MSA patients (39, 40). The outstanding question still remains as to how the same polypeptide chain can produce such a vast number of polymorphic structures. While there are significant structural differences, some features of α-syn fibrils are conserved. All fibrils are formed from a twisting pair of protofilaments with the exception of a H50Q polymorph, which is composed of a single filament. A kernel motif of a bent β-arch appears in all structures. Also, at least one inter- or intramolecular salt bridge between a Lys and Glu is revealed in each structure (24, 30–38, 40), which is not surprising given that there are numerous possibilities for salt bridges between the 14 Lys, 8 Glu, and 2 Asp residues located throughout the first 100 residues in the sequence (Fig. 1A and SI Appendix, Fig. S1). Generally, residues between 37 and 97 constitute the fibril core with a few exceptions that involve additional residues in the N terminus, which include phosphorylated Y39 fibrils with an extended core of 1–100 (34) and two polymorphs of 1–140 showing interactions of N-terminal β-strands (residues 14–24) (30). Fibrils derived from brains of MSA patients also indicate additional involvement of the N-terminal region extending to residue 14 (40). Due to the contribution of N-terminal residues in these structures and the fact that C-terminal truncations resulted in modest conformational changes, we hypothesize that N-terminal residues play a greater role in influencing fibril structure.Open in a separate windowFig. 1.Aggregation of ΔN-α-syns. (A) Schematic representation of α-syn primary sequence (residues 1–140), showing basic (blue) and acidic (red) residues. Underlined regions correspond to truncations used in this study: 14‒140 (blue), 36‒140 (magenta), and 41‒140 (green). (B and C) Comparison of aggregation kinetics monitored by ThT fluorescence at 37 °C. [α-Syn] = 35 µM (B) and 70 µM (C) with [ThT] = 10 µM in 20 mM NaPi, 140 mM NaCl, pH 7.4. The solid line and shaded region represent the mean and SD, respectively (n ≥ 4). Representative TEM images of (D) 1‒140, (E) 14‒140, (F) 36‒140, and (G) 41‒140 were taken at 35 µM. Different fibril polymorphs observed are noted. Additional fields of view are shown in SI Appendix, Figs. S3–S5.Here, we sought to understand the role of the N terminus in α-syn fibril formation by removing different N-terminal residues and evaluating their effects on aggregation kinetics, fibril structure, and propagation. Three ∆N-terminal constructs (14‒140, 36‒140, and 41‒140) have been examined, in which the first 13-, 35-, and 40-residues in the N terminus were deleted (Fig. 1A). We specifically chose these sites based on the locations of native Gly residues, which allows us to generate native sequences (i.e., no overhang) upon Tobacco Etch Virus (TEV) protease cleavage of the hexahistidine affinity tag, which facilitates facile protein purification. All three ∆N-α-syn exhibited different aggregation kinetics and distinct fibril ultrastructural features as determined by thioflavin-T (ThT) fluorescence and transmission electron microscopy (TEM), respectively. In cross-seeding experiments, both fibrillar 36‒140 and 41‒140 did not seed the full-length (1‒140) protein, while 14‒140 fared better but less efficient than self-seeding, supportive of the significant impact of removing N-terminal residues in fibril structure. The reverse reaction involving full-length seeds showed that fibril formation of 14‒140 and 36‒140 but not 41‒140 could be accelerated. This observation is explained by the fibril structure adopted by 41–140, which was determined by cryo-EM to an overall resolution of 3.2 Å. Unlike any currently known α-syn structure, the amyloid core is formed by two asymmetric protomers with different amino acid chain lengths, adopting an extended β-hairpin (E61‒D98) and the bent β-arch kernel (E46‒K96) with a large nonpolar interfilament interface (442 Ų) stabilized by an intermolecular salt bridge between K80 and E83. Collectively, these results establish the important role of N-terminal residues in fibril formation and structure.     

Relationship between Structure and Antibacterial Activity of α-Aminophosphonate Derivatives Obtained via Lipase-Catalyzed Kabachnik−Fields Reaction

We reported a new method dealing with the synthesis of novel pharmacologically relevant α-aminophosphonate derivatives via a lipase-catalyzed Kabachnik−Fields reaction with yields of up to 93%. The advantages of this protocol are excellent yields, mild reaction conditions, low costs, and sustainability. The developed protocol is applicable to a range of H-phosphites and organic amines, providing a wide substrate scope. A new class of α-aminophosphonate analogues possessing P-chiral centers was also synthesized. The synthesized compounds were characterized on the basis of their antimicrobial activities against E. coli. The impact of the various alkoxy groups on antimicrobial activity was demonstrated. The crucial role of the substituents, located at the aromatic rings in the phenylethyloxy and benzyloxy groups, on the inhibitory action against selected pathogenic E. coli strains was revealed. The observed results are especially important because of increasing resistance of bacteria to various drugs and antibiotics.     

Synthesis and Characterization of Single-Phase α-Cordierite Glass-Ceramics for LTCC Substrates from Tuff

Single-phase α-cordierite glass-ceramics for a low-temperature co-fired ceramic (LTCC) substrate were fabricated from tuff as the main raw material, using the non-stoichiometric formula of α-cordierite with excess MgO without adding any sintering additives. The sintering/crystallization behavior and the various performances of dielectric properties, thermal expansion, and flexural strength of the glass-ceramics were detected. The results indicated that only single-phase α-cordierite crystal was precipitated from the basic glass sintered at the range 875–950 °C, and μ-cordierite crystal was not observed during the whole sintering-crystallization process. The properties of glass-ceramics were first improved and then deteriorated with the increase in tuff content and sintering temperature. Fortunately, the glass-ceramics sintered at 900 °C with 45 wt.% tuff content possessed excellent properties: high densify (2.62 g∙cm⁻³), applicable flexural strength (136 MPa), low dielectric loss (0.010, at 10 MHz), low dielectric constant (5.12, at 10 MHz, close to α-cordierite), and suitable coefficients of thermal expansion (CTE, 3.89 × 10⁻⁶ K⁻¹).     

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β₂-adrenergic receptors (β₂AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β₂AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β₂AR actions favorable for treating obstructive lung disease.

Most G protein–coupled receptors (GPCRs) are now recognized as multisignal transducers (1, 2). Early concepts of agonist–receptor interactions were based on the idea that there was a single “active” receptor conformation induced by the binding of any agonist, resulting in an interaction with the heterotrimeric G protein and a universal, singular signal. Generally, the α-subunit of the G protein, upon its dissociation, was considered the primary activator (or inhibitor) of the effector, resulting in the intracellular signal. Subsequently, it became clear that multiple signaling outcomes from activation of a given GPCR can occur from a single agonist due to specific molecular determinants of the receptor triggering independent mechanisms (3–5). As these multiple functions were being identified, it was apparent that agonists with different structures could act at a given receptor to preferentially activate one signal with minimal engagement of others, a property later termed signal biasing (6–8). Biased agonists, then, could represent important advantages over nonbiased agonists due to this signal selectivity, activating a specified therapeutic pathway while minimally evoking unnecessary or deleterious signaling. The pathway selectivity of biased agonists is thought to be established by the stabilization of specific conformation(s) of the agonist–receptor complex via a set of interactions that differ from those of unbiased (also called balanced) agonists (9–12). While it is conceivable that small modifications of established cognate agonists might yield such specialized signaling, significant deviation from common agonist structures may be necessary to meet this goal (13).The signals/functions of a given GPCR that might be sought for selective activation are defined by the cell type, disease, and desired final physiologic function. In asthma and chronic obstructive pulmonary disease (COPD), active human airway smooth muscle (HASM) cellular contraction limits airflow, representing a major cause of morbidity and mortality. β₂-adrenergic receptors (β₂ARs) expressed on HASM cells are the targets for binding of therapeutically administered β-agonists, which relax the cells via a cyclic adenosine monophosphate–mediated mechanism (14). β-agonists are used for treating acute bronchospasm as well as for long-term prevention. However, the HASM bronchodilator response to acute β-agonist is attenuated by receptor desensitization (15), with typical treatments of humans, or isolated HASM cells, leading to a loss of receptor function over time (16–18), clinically termed tachyphylaxis.Agonist-promoted desensitization of β₂AR (and other GPCRs) is due to partial uncoupling of the receptor to the G protein, which is initiated by phosphorylation of intracellular Ser/Thr residues of the receptor by G protein–coupled receptor kinases (GRKs) (19, 20). The GRK-phosphorylated β₂AR recruits β-arrestin1 or β-arrestin2 to these receptors, with subsequent interactions that appear to compete with the receptor for its binding to the Gα subunit, thus attenuating the intracellular response (11, 21). Such competition has been strongly inferred for the β₂AR (22, 23) and is compelling for rhodopsin–arrestin interactions (24). In addition, β-arrestin binding to GPCRs can initiate receptor internalization and other events such as receptor activation of ERK1/2 (25) through its multiprotein adapter functions. Thus β-arrestin engagement can be considered an early “second signal” of the β₂AR as well as a desensitization initiator for attenuating the Gs signal. An agonist that is biased toward Gαs coupling (cAMP production and airway smooth muscle [ASM] relaxation) and away from β-arrestin binding (desensitization) would be desirable in treating obstructive lung diseases, since efficacy would not be attenuated acutely, nor would tachyphylaxis be experienced from extended treatment. While biased agonists favoring either G protein or β-arrestin (6) signaling have been described for some GPCRs (such as μ-opioid and type 1 angiotensin II receptors), Gαs biasing has not been apparent from most studies with catecholamine-like compounds for the β₂AR. Thus, we have little information as to whether the two β₂AR pathways can be differentially activated in a selective manner by an efficacious agonist, nor is it apparent from a structural standpoint what strategy might be employed to design agonists biased in this manner for this receptor.In order to find this type of biasing for the β₂AR, we screened a 40-million-compound scaffold ranking (SR) library that was agnostic to known β₂AR agonist structures. We found a scaffold in which substitutions of certain R groups led to individual compounds that are apparently Gαs-biased agonists for β₂AR with no apparent engagement of β-arrestin in model systems. Additional studies in HASM cells revealed a lack of tachyphylaxis of the relaxation effect by the lead compound compared with the most widely utilized β₂AR agonist, albuterol. The structure of this biased agonist is very different from that of catecholamine-like agonists. To ascertain the mechanism that may underlie this biased activity, we used structural modeling and molecular simulations and studied homologous compounds with different R groups and receptor mutagenesis to predict the interaction sites with the activated β₂AR. Such studies uncovered distinct structural characteristics that may be responsible for the biasing effect.     

Human Adenovirus Type 26 Induced IL-6 Gene Expression in an αvβ3 Integrin- and NF-κB-Dependent Manner

The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was the first adenovirus-based vector to receive marketing authorization from European Commission. HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such, represent a highly valuable tool for fighting infectious diseases. Despite well-described immunogenicity in vivo, the basic biology of HAdV26 still needs some refinement. The aim of this study was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and then investigate the underlying molecular mechanism. The expression of studied genes and proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines and chemokines, namely IL-6, IL-8, IL-1β, and TNF-α, with the most pronounced difference shown for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced IL-6 gene expression is αvβ3 integrin dependent and NF-κB mediated. Our findings provide new data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained within this research increases our current knowledge of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-based vaccine vectors.     

Behaviors of Micro-Arcs,Bubbles, and Coating Growth during Plasma Electrolytic Oxidation of β-Titanium Alloy

The plasma electrolytic oxidation (PEO) of a titanium alloy, Ti-15V-3Cr-3Sn-3Al, was performed to develop mechanical applications by improving the tribological characteristics. The behaviors of micro-arcs, bubbles, and coating growth during the PEO process were investigated under three different operating conditions, constant voltage (CV) operation, constant current operation (CC), and short treatment time (ST) operation, to control the surface structure and function by the PEO process. A low friction coefficient was achieved by CV operation at 500 V and by CC operation at 3.0 kA/m². The maximum coating thickness of 6.88 μm was achieved by CV operation at 500 V and 60 s. From the observation of micro-arcs, bubbles, and discharge craters by ST operation, the minimum discharge diameter of the micro-arc was 8 μm, and the discharge craters had a discharge pore size of 0.3 μm in diameter in the center with a petal-shaped burr around the discharge pore. During the PEO process, no bubble bursts around the micro-arcs and no backfilling of the discharge pores by the ejected materials were observed. Thus, the discharge pores remain a porous structure in the PEO coating for Ti. The utilization efficiency of the total charge density by CV operation above 300 V was lower than that by the conventional anodization process. The utilization efficiency of total charge density by CC operation was higher than that by the conventional anodization process.     

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo

GBA1 mutations that encode lysosomal β-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson’s disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Gaucher disease (GD) is a lysosomal storage disorder caused by loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase). GCase degrades glycosphingolipids (GSLs), including glucosylceramides (GluCers), into glucose and ceramide, and GCase mutations result in the accumulation of GluCer in lysosomes of various tissues. Heterozygote carriers of the same loss-of-function GCase mutations are estimated to be at 5- to 10-fold higher risk for developing Parkinson’s disease (PD) or Lewy body dementia (1). In GD, significant variability exists in the clinical and pathological presentation, resulting in three main GD subtypes (2). Type 1 GD is characterized by visceral abnormalities, including enlarged liver and spleen and bone marrow dysfunction, leading to thrombocytopenia but without neurodegeneration and α-synuclein (α-syn) pathology (3). Types 2 and 3 demonstrate similar visceral symptoms but with additional extensive neuronal loss, α-syn pathology in the form of classical Lewy bodies, and neurological dysfunction (3, 4). As life expectancy of type 1 GD has increased because of enzyme replacement therapy, a higher percentage of patients develop PD symptoms with age (5), suggesting that aging could contribute to the penetrance of GBA1 mutations. The dramatic phenotypic heterogeneity suggests that GD is not a simple, monogenic disease but a complex disorder that is influenced by both genetic and nongenetic modifiers. Although the factors that contribute to clinical and pathological variability in GD are not known, genetic modifiers have been identified that associate with GD severity, including CLN8 and SCARB2 (6, 7). Within PD patients that harbor GBA1 mutations (GBA-PD), the search for genetic modifiers has shown that synergism may exist with the SNCA gene that encodes α-syn and CTSB that encodes lysosomal cathepsin B (8). Variants in lysosomal cathepsins could influence the severity of α-syn accumulation, since, under physiological or pathological conditions, α-syn can be degraded by the lysosome (9–11) and is a direct substrate of cathepsin B and L (12).An additional factor that may contribute to phenotypic variability in GD is the accumulation of specific GluCer subtypes with particular acyl chain lengths. GluCer and other GSLs exist as a family of lipid isoforms differentiated by the length of the N-acyl fatty acid moiety linked to the sphingoid base. GluCer chains range from C14 to C26 in the brain; however, C18 and C24:1 are the predominant species (13). Studies of neuronopathic GD (nGD) brain or mouse models showed intraneuronal accumulation of multiple GluCer species that correlated with neuroinflammation (14–19), and some cases demonstrate selective accumulation of long-chain GluCers in nGD (20). Our recent work in PD patient midbrain neurons showed that inhibition of wild-type (wt) GCase, caused by α-syn, resulted in the selective accumulation of long-chain-length GluCers, including C22 and C24:1, while C14, 16, and C18 were unchanged (21). Together, these data indicate that GluCer accumulation plays an important role in neurodegeneration induced by GBA1 mutations; however, the specific contributions of distinct GluCer species have not been examined.Here, we extend our studies on the role of GSLs in α-syn aggregation to further define conditions that are required to induce pathology and neurological dysfunction. We previously showed that α-syn exists as monomers and high–molecular weight (HMW) oligomers under physiological conditions in human midbrain cultures (22). In vitro, we found that GluCer mildly induced aggregation of α-syn monomers but primarily acted on physiological oligomers to convert them into toxic oligomers and fibrillar inclusions (22). α-syn accumulation can be prevented or reversed by reducing GSLs with GluCer synthase inhibitors (GCSi) in both GD and PD patient cultures, as well as in mouse models (22–24). While this work suggests a close relationship between GCase function and α-syn pathology, additional factors must exist that create a permissive environment for α-syn accumulation. Indeed, studies that used newborn mice or embryonic primary neuron cultures treated with the GCase inhibitor, conduritol beta epoxide (CBE), have shown no changes in α-syn despite reduced GCase activity (25–27). However, other studies that use matured neuron cultures, neuronal cell lines, or adult mice have shown that CBE dramatically induces α-syn aggregates (22, 28–31). We used an in vivo GD model and induced pluripotent stem cell (iPSC)–derived patient midbrain cultures to identify specific conditions that are required to induce α-syn pathology, providing possible explanations for the variable neurological penetrance in patients that harbor GBA1 mutations.     

Wild-type GBA1 increases the α-synuclein tetramer–monomer ratio,reduces lipid-rich aggregates,and attenuates motor and cognitive deficits in mice

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson’s disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient–derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer–monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD–causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)–human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

GBA1 gene mutations in Gaucher’s disease carriers are recognized as the most important risk factors for developing Parkinson’s disease (PD), since large multicenter patient cohorts identified GBA variants in PD, including in ∼3% of sporadic PD patients and up to ∼15% of the Ashkenazi Jewish population with PD (1). Homozygous and heterozygous GBA1 mutation carriers display a similar risk (∼20%) of developing PD (2). GBA1 mutations can impact the activity of its gene product, the lysosomal lipid metabolism enzyme glucocerebrosidase (GCase), leading to changes in cellular lipid content and lipid membrane morphologies (3, 4). Clinically, PD patients with GBA1 mutations are largely indistinguishable from the idiopathic form. Both populations exhibit widespread α-synuclein (αS)+ Lewy bodies (LBs), including in the hippocampus and other brain regions, and these are associated with motor deficits and cognitive decline (2). PD-GBA1 mutation carriers are at a greater risk of cognitive impairments, and this finding is consistent with a higher incidence of GBA1 mutations in =DLB patients (5, 6). Recent morphological analyses of “sporadic” PD brain tissues have revealed that Lewy-type inclusions also contain substantial amounts of lipid-rich membranes and vesicles, including lysosomes (7). Additional evidence for the role of GCase in αS homeostasis has been generated in mouse studies and in GBA1-mutant neural cells, suggesting increased accumulation of αS secondary to different pathogenic GBA1 mutations (8–11).Accumulating evidence from our laboratory (12–14) and others (15–18) shows that αS normally occurs in a dynamic equilibrium between helically folded tetramers and “natively unfolded” monomers. Regarding the relevance of αS tetramers to disease, we found that all familial PD (fPD)–causing αS mutations decrease the physiological tetramer–monomer (T:M) ratio and some induce cytoplasmic inclusions and neurotoxicity in human (hu) and rodent cell culture (13). Supporting these findings, neurons harboring PD-causing GBA1 mutations shifted endogenous wild-type (wt) αS tetramers to monomers that lead to abnormal phosphorylated serine 129 αS (pS129) + αS accumulation (18), indicating lipid metabolism can impact physiological αS homeostasis. Mechanistic studies have shown that saturated fatty acids (SFAs) stabilize normal tetramers, while unsaturated FAs, such as oleic acid, decrease the T:M ratio (19, 20). Accordingly, decreasing stearoyl-CoA desaturase (SCD) activity, the rate-limiting enzyme for generating monounsaturated (MU) FA, decreases αS+ neuronal inclusions in yeast, rat cortical neurons, hu wt, fPD E46K–induced neurons, and in 3K cell culture models (19–21).Our recent approach to treating hu wt or 3K αS mutant mice with SCD inhibitors showed that the prolonged increases in the T:M ratio can reduce excess triacylglycerides (TAGs), lipid droplets (LDs) (rich in TAGs), and pS129 αS+ aggregates, aiding multiple PD motor phenotypes (22). Intriguingly, overexpressing hu wtGBA increased the αS T:M ratio in Gaucher’s GBA1-mutant neuronal culture (18).Whether early transduction and prolonged increase of hu wtGCase can enhance αS T:M homeostasis in vivo has yet not been examined. To begin investigating this question, we used the tetramer-abrogating “3K” αS mutant mouse line that is a biochemical amplification of the E46K mutation-causing PD. The 3K mutation shifts the normally aggregation-resistant αS tetramers (12) to increased levels of monomers that then cluster with vesicle membranes and form sizeable aggregates, thereby producing multiple PD-like motor phenotypes by the age of 6 mo (23). The Thy1.2 promotor that drives the 3K transgene reaches stable expression from postnatal day 7 onwards (24), thereby enabling us to study whether GBA1 effects the onset of αS dyshomeostasis in mouse brain when injecting it into 3K neonates. Here, we transduced an adeno-associated virus (AAV)–wtGBA1 vector by intracerebroventricular (ICV) injections in 3K and control littermate pups at P1 and then, 6 mo later, performed motor and cognitive testing and examined the brains for αS species, GCase activity, lysosomal abnormalities, and lipid aggregation patterns.     

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species

α-synuclein aggregation is present in Parkinson’s disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

Aberrant aggregation of α-synuclein (α-syn) into amyloid fibrils and subsequent accumulation into intracellular inclusions is a hallmark of neurodegenerative disorders such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy (1–3). In these diseases, soluble α-syn monomers misfold and self-assemble, forming small oligomeric species that retain the highly disordered structure of the monomeric state (4). These species are rather unstable and can undergo structural rearrangements, including a gain in β-sheet structure that generates more stable species (4, 5). β-structured oligomers can grow further through monomer addition or self-association, finally giving rise to well-defined amyloid fibrils (4–6). Despite the controversial evidence about the relationship between the different species that populate the aggregation process and cellular toxicity, the prevalent view is that both intermediate oligomers and small fibrils are neurotoxic (7). Due to their abnormal interactions with cellular components, certain types of oligomers are key pathogenic agents in the development of the disease (8–10). In particular, they can disrupt membranes, induce oxidative stress, dysregulate calcium homeostasis, cause mitochondria dysfunction, or impair the proteasome system (11). Furthermore, α-syn oligomers have been implicated in the spreading of the disease, as these aggregates can be transmitted between cells (12, 13). Small fibrils have also been related to intercellular spreading and propagation of neurodegeneration (14–18). In contrast, large amyloid aggregates are believed to be relatively inert, as their highly ordered packing and slow diffusion reduces undesired interactions with cellular components. Even so, large aggregates can generate intermediate species that contribute to cytotoxicity through secondary processes such as fragmentation or nucleation on the aggregate surface (19, 20).To counteract the toxic effect of protein aggregates, cells have evolved a sophisticated protein homeostasis network that coordinates protein synthesis, folding, disaggregation and degradation (21). This network is composed of the translational machinery, molecular chaperones and cochaperones, the ubiquitin-proteasome system, and the autophagy machinery. The way this network tackles amyloid aggregates remains poorly understood. It has been previously reported that the constitutive human Hsp70 (Hsc70) in collaboration with its Hsp40 cochaperone (Hdj1 or DnaJB1) slowly disassembles preformed α-syn fibrils (22). This activity was further stimulated by adding the NEF Hsp110 (Apg2). HspB5, a small heat shock protein also known as αB-crystallin, potentiated α-syn fibril disassembly by the ternary chaperone mixture. Although this chaperone combination was able to disaggregate fibrils, they did it in a timescale of weeks through a depolymerization process. Only when Hsp104, a yeast representative of the Hsp100 family able of fragmenting fibrils, was added to the mixture, disassembly occurred within hours (22). The lack of Hsp104 homologs in metazoans questioned whether this activity was physiologically relevant in humans. A later study revealed that a chaperone complex composed solely of members of the Hsp70, Hsp40, and Hsp110 families was able to efficiently reverse α-syn amyloid fibrils through both fragmentation and depolymerization, generating smaller fibrils, oligomers, and, ultimately, monomers (23). Despite the importance of this emerging disaggregase functionality, its mechanism of action remains largely unknown. Recently, the same chaperone mixture has been reported to also disaggregate tau and Htt fibrils (24–26), pointing to this Hsp70-based machinery as a potential human amyloid disaggregase.The two-fold aim of this work is, firstly, to test whether human disaggregase remodels with the same efficiency the different aggregates that populate the complex process of amyloid formation and, secondly, to shed light on the key mechanisms involved in the disassembly of amyloids. We show that the human disaggregase system disassembles toxic oligomers and short fibrils much better than large, less toxic fibrils, and that it does so by an enhanced destabilization of the small aggregated forms. Explicitly, fibril disassembly involves destabilization of the fibril ends and unzipping of the protofilaments, which allow depolymerization. The fast propagation of protofilament depolymerization toward the opposite fibril end is consistent with entropic pulling forces exerted by Hsc70 upon binding the fibril surface.     

Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Synucleinopathies, including Parkinson’s disease (PD), are associated with the accumulation of intracellular neuronal aggregates termed as Lewy bodies and Lewy neuritis, which contain high concentration of the protein α-synuclein (α-Syn) in an aggregated state (1, 2). The disease-relevant role of α-Syn is further highlighted by mutations in the α-Syn gene (SNCA) causing familial PD [i.e., A30P (3), E46K (4), H50Q (5), G51D (6), A53E (7), and A53T (8)] and the duplication or triplication of the SNCA leading to early-onset PD in affected families (9, 10). α-Syn is a 140-residue intrinsically disordered protein (IDP) in solution (11) but adopts a helical structure in the presence of acidic lipid surfaces (12, 13). The positively charged N terminus (residues 1 to 60) is rich in lysine residues and contains KTKEGV binding repeats associated with vesicle binding (14). Moreover, the N-terminal domain includes all known SNCA familial PD mutations. The central region (residues 61 to 95) defines the non-amyloid-β component (NAC) (15), which is essential for α-Syn aggregation (16), while the C terminus (residues 96 to 140) is highly negatively charged.In vitro, α-Syn forms polymorphic amyloid fibrils (17–19) with unique arrangements of cross-β-sheet motifs (20–22). When injected into model animals, these fibrils induce a PD-like pathology (23) where the aggregation pathway of α-Syn plays a key role in the development of the disease (24). A detailed analysis of the aggregation kinetics of α-Syn into amyloids is therefore important toward understanding the toxic mechanisms relevant for synucleinopathies.Amyloid formation of α-Syn is very sensitive to solution conditions, including pH (25), temperature (26), and salt concentration (27). It further requires the presence of an air–water interface (28) or negatively charged lipid membranes (29) for which α-Syn has a high affinity. Previous studies suggest that amyloid fibril growth of α-Syn occurs via a nucleation-dependent polymerization reaction (30). Following a fairly slow primary nucleus formation, α-Syn fibrils are elongated by addition of single monomers. In a next step, the amyloid fibrils multiply by fragmentation or can catalyze the formation of new amyloids from monomers on their surface—a process known as secondary nucleation that was first described for sickle cell anemia 40 y ago (31). Fragmentation and secondary nucleation critically depend on the fibril mass and accelerate the aggregation kinetics (30). In the case of α-Syn aggregation under quiescent condition fragmentation does not exist and only the described secondary nucleation process occurs. While detailed kinetic experiments showed no significant secondary nucleation at pH 7, it strongly contributes at pH values lower than 6 (25, 30). However, mechanistic or structural information of the secondary nucleation process in α-Syn aggregation has been lacking so far.In this study we investigated the structural properties of α-Syn monomer–fibril interactions by NMR and electron paramagnetic resonance (EPR) spectroscopy. Our results provide insights into how monomeric α-Syn transiently interacts in vitro via its positively charged N terminus with the negatively charged C-terminal residues of the α-Syn fibrils, giving detailed insights into the mechanism of the secondary nucleation process.     

A Review of Irradiation Damage and Effects in α-Uranium

Understanding irradiation damage and effects in α-uranium (α-U) is critical to modeling the behavior of U-based metallic fuels. The aim of this review is to address the renewed interest in U-based metallic fuels by examining the state-of-the-art knowledge associated with the effect of irradiation on the microstructure, dimensional changes, and properties of α-U. We critically review the research progress on irradiation-induced growth and swelling, the enhancement of plastic flow and superplasticity by irradiation, and the effect of irradiation on thermal and electrical properties of α-U. Finally, we outline the research directions that require advancements, specifically the need to carry out fundamental research on several of the less understood mechanisms of irradiation damage and effects in α-U.     

Calcineurin regulates the stability and activity of estrogen receptor α

Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser²⁹⁴ in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser²⁹⁴ promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser¹¹⁸ by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to β-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser²⁹⁴ and Ser¹¹⁸. Finally, the expression of the calcineurin A–α gene (PPP3CA) was associated with poor prognosis in ER-α–positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α–positive breast cancer.

Estrogen receptor α (ER-α) plays a central role in the proliferation of breast cancer cells by increasing the expression of oncogenes, such as those encoding cyclin D1 and c-Myc (1). The expression and activity of ER-α are increased in >70% of breast cancer cases, and the receptor is targeted by drugs such as tamoxifen (2, 3). A substantial proportion of ER-α–positive breast cancer cells become resistant to anti‐estrogens, however, resulting in the progression of the disease. The mechanisms by which the cancer cells acquire resistance to these agents include the generation of splice variants of ER-α, the mutation of the ER-α gene (ESR1), and changes in stability of the ER-α protein (4).Increased protein stability appears to be a key contributor to the up-regulation of ER-α in breast cancer. The ubiquitination of ER-α is one mechanism responsible for ER-α degradation. Several E3 ligases that mediate the degradation of ER-α have been identified and include E6-associated protein (E6AP) (5), carboxyl terminus of Hsp70-interacting protein (CHIP) (6), breast cancer type 1 (BRCA1) (7), BRCA1-associated RING domain 1 (8), S phase kinase–associated protein 2 (SKP2) (9), and mouse double minute 2 homolog (10). On the other hand, other E3 ligases—such as RING finger protein (RNF) 31, shank-associated RH domain–interacting protein, and RNF8 (11–13)—have been shown to promote ER-α signaling by stabilizing ER-α protein.The residues Lys³⁰² and Lys³⁰³ of ER-α are targeted for ubiquitination (14). The ubiquitination of ER-α is associated with its phosphorylation, with several kinases such as cyclin-dependent kinase (CDK) 11 (15), Src (5), protein kinase C (16), p38 mitogen-activated protein kinase (9), and extracellular signal–regulated kinase 7 (17) having been shown to phosphorylate the protein. The phosphorylation of ER-α at Ser²⁹⁴ has thus been related to its ubiquitination by SKP2 (9), with the Ser²⁹⁴-phosphorylated form of ER-α being a preferred substrate for ubiquitination by SKP2 in vitro. However, the expression level of ER-α was found to be unaltered in cells depleted of SKP2, suggesting that other E3 ligases may contribute to the degradation of ER-α subsequent to its phosphorylation at Ser²⁹⁴.Calcium is an important regulator of signaling pathways that control oncogenesis and cancer progression, and Ca²⁺ signaling has been linked to signaling by ER-α. β-estradiol (E2) has been shown to induce rapid Ca²⁺ influx in cells, and the Ca²⁺-binding protein calmodulin interacts with ER-α, increases its stability, and modulates E2-regulated gene expression (18). Calcineurin is a Ca²⁺/calmodulin-activated serine–threonine phosphatase that plays a major role in the regulation of immediate cellular responses and gene expression by Ca²⁺ signaling (19). It is also a target of immunosuppressive drugs administered in clinical practice, such as cyclosporine A and FK506. Calcineurin is composed of two subunits: a catalytic subunit, designated calcineurin A, that is encoded by three genes (PPP3CA, PPP3CB, and PPP3CC), and a regulatory subunit, designated calcineurin B, that is encoded by two genes (PPP3R1 and PPP3R2).In the present study, we found that calcineurin plays a previously unrecognized role as a positive regulator of the stability and activity of ER-α in breast cancer cells by mediating its dephosphorylation at Ser²⁹⁴, as well as the activation of mechanistic target of rapamycin complex 1 (mTORC1) and the consequent phosphorylation of ER-α at Ser¹¹⁸, respectively. Furthermore, a high-expression level of PPP3CA was associated with poor prognosis in a subset of breast cancer patients, suggesting that the selective inhibition of calcineurin might be an effective approach to the treatment of ER-α–positive breast cancer.